This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. S-(2-succinyl)cysteine (2SC) is the product of a Michael addition reaction between the Krebs cycle intermediate, fumarate, and thiol groups in proteins - a process termed succination of protein. This novel post-translational modification increases >10 fold in adipocytes grown in high glucose medium, in muscle of type 1 diabetic rats, and in adipose tissue of type 2 diabetic (db/db) mice. Over 60 2SC-protein spots have been detected by 2D-PAGE and western blotting with anti-2SC antibody in adipocyte and adipose tissue proteins, including adiponectin, contractile proteins, heat shock proteins, enzymes and regulatory proteins. 2SC is considered an early biomarker of mitochondrial and oxidative stress in diabetes and may also have a role in signal transduction in response to glucotoxicity in diabetes or pre-diabetic states, such as obesity and metabolic syndrome. The goal of this project is to define the 2SC proteome in adipose tissue of db/db mice and thereby to gain insight into the role of this chemical modification of protein in the alteration of metabolism in diabetes. The project will employ resources at PNNL for high resolution two-dimensional LC separations of peptides in conjunction with identification by tandem mass spectrometry (MS/MS) in order to develop a database of all possible 2SC-peptides from incubation of adipocyte proteins with high fumarate concentration. Subsequent high resolution LC-FTICR MS experiments together with a double isotope tagging technique will be used for comparative analysis of adipose tissue from control and diabetic mice. The information obtained and methods developed in these studies will be used as preliminary data for NIH research proposals on the role of succination in development of diabetes and its retinal, renal and vascular complications. Parallel applications could include analysis of the scope and extent of other chemical modifications of protein in diabetes, including oxidative, glycoxidative and lipoxidative damage to proteins.